Abstract

Haemophilus influenzae is an important human pathogen, responsible for respiratory infections, such as otitis media, bronchitis and epiglottitis, as well as invasive disease. Despite being the first free-­‐living organism to have its whole genome sequenced, there have been only a few published studies investigating its transcriptional profile using next-­‐generation sequencing (NGS). The work presented in this thesis aimed to use NGS to improve the understanding of how H. influenzae behaves during natural infection and to identify novel RNA structures with potentially important roles in pathogenesis.

The whole transcriptome of H. influenzae during infection-­‐relevant conditions was analysed using high-­‐throughput RNA sequencing. For the first time, the transcriptional profile of H. influenzae during stationary phase and nutritional stress was determined on a whole-­‐genome scale. Differential gene expression analysis of an invasive strain, R2866, and a laboratory strain, Rd KW20, revealed differences in their transcriptional response, particularly during oxidative stress and iron starvation. Importantly, a new systematic and robust bioinformatic tool, "toRNAdo", was developed to identify non-­‐coding RNA elements from the bacterial transcriptomic data. It enabled discovery of a repertoire of novel putative intergenic and antisense non-­‐coding RNAs in H. influenzae. In addition, the first fully sequenced genome of a free-­‐living organism, the Rd KW20 strain of H. influenzae, was re-­‐sequenced and re-­‐ annotated for the first time. This enabled identification of multiple nucleotide-­‐ level differences between original and re-­‐sequenced genomes of Rd KW20.

The work presented here facilitates future characterisation of novel RNA elements, with potentially important regulatory roles in pathogenesis in H. influenzae, and has implications for defining a model bacterial strain. Importantly, the findings present significant insight into the pathogenic lifestyle of H. influenzae. They provide the basis for further work, where novel vaccine and antibiotic targets may get developed.